This application requests continued funding for the TRiP, a highly successful in vivo Drosophila functional genomics platform at Harvard Medical School. To date we have generated more than 17,000 RNAi and CRISPR fly stocks for the research community. We propose to expand the TRiP resource of versatile and transformative transgenic tools for gene activation, repression and genome engineering. The goals of this resource, focused on the 3017 fly genes orthologous to genes known or suspected to be associated with human diseases, are to: (1) generate shRNA fly stocks for RNAi that address remaining gaps in the collection, such as genes not yet covered or reagents identified by the community as ineffective; (2) generate sgRNA stocks for CRISPR knock out (CRISPRko) and CRISPR activation (CRISPRa); (3) Build a toolkit of reagents that combine GAL4/UAS and a second binary expression system (LexA/lexAop or QF/QUAS) to facilitate RNAi and/or CRISPR control of gene expression in two different tissues independently; and (5) evaluate whole animal as well as tissue-specific loss-of-function (LOF) and gain-of-function (GOF) phenotypes associated with the new lines and curate information on the quality of individual RNAi and sgRNA lines in our ?RSVP Plus? phenotype database. The result of these efforts will be a tremendous resource of fly stocks, distributed to the community by the Bloomington Drosophila Stock Center, which will allow researchers to easily knock down, knock out, or activate genes covered by the collection. When combined with the wide array of GAL4 lines, and our proposed new collection of LexA/QF lines, this genetic toolkit will allow researchers to modulate gene expression in any given stage and in multiple tissues simultaneously.